Advanced imaging and physiology resources for common use
The goal of the Multiphoton Imaging Core is to provide instrumentation for analyzing protein localization, protein dynamics, and protein-protein interactions with high resolution. This facility also allows users to perform time-lapse imaging of multiple fluorophores in living cells and tissues, and to combine high resolution imaging of fluorescently tagged proteins or ion indicator dyes with electrophysiological monitoring of electrical activity.
The Multiphoton Imaging Core promotes interactions among a diverse group of neuroscientists at Johns Hopkins University. This has facilitated the investigation of key issues in basic and clinical neuroscience, as illustrated in our List of Publications of work supported by the MPI Core.
The MPI Core is funded by an NINDS Core Center Grant (P30 NS050274: “JHU Center for Neuroscience Research”). Services provided by this, the other two Cores funded by this grant, and the Machine Shop, are available to three groups of investigators: (1) the thirteen Primary Center Investigators who are co-PIs of this P30 grant; (2) NINDS-funded investigators at The Johns Hopkins School of Medicine; and (3) on a case by case basis, other non-NINDS-funded neuroscience investigators at JHU.
All applications for use of Core facilities are carefully evaluated by Core personnel and directors, and services are allocated based on availability and guidelines that govern this NINDS Core funding mechanism. Questions regarding eligibility to utilize Core Services can be addressed to the MPI Core Director (Dwight Bergles, firstname.lastname@example.org) or the P30 Center Director (Alex Kolodkin, email@example.com). We can also provide expertise and resources for performing pilot experiments for a broad range of research.
The Multiphoton Imaging Core is located in Room 1008A, Preclinical Teaching Building, 725 North Wolfe Street, on the Johns Hopkins University East Baltimore campus.
A great deal of expertise is available to users of the Multiphoton Core Facility. Michele Pucak, PhD (MPI Core Manager) has experience in single and multiphoton imaging, immunohistochemistry, maintenance of animals for in vivo imaging, and electrophysiological techniques.
The MPI Core Director, Dr. Dwight Bergles, has extensive expertise in imaging and electrophysiological techniques. In addition, the many users of the facility each bring unique knowledge and experience that can be leveraged for the particular experimental needs of investigators.
The facility contains three confocal microscope systems that allow high resolution imaging of labeled cell components in three-dimensional space. The first system consists of a Zeiss LSM 510 motorized upright microscope, equipped with a META scanning module, two non-descanned detectors for multi-photon work, and a motorized stage for time lapse imaging. The LSM 510 can be configured with the META detector plus two traditional detectors for fluorescent or reflected light imaging. The META detector can also be used as a unique spectral detector or a conventional "band-pass" detector.
All three detectors can be recorded and displayed simultaneously. This system employs a PC-based user interface to control the microscope, laser excitation, scanning and image acquisition, and is capable of digitally capturing up to 12 bit 2K x 2K pixel images, while the AIM LSM software controls image data acquisition and processing. The system can perform Fluorescent Recovery after Photobleaching (FRAP), Fluorescence Resonance Energy Transfer (FRET), and quantitative co-localization.
The second system is a Zeiss AxioExaminer upright microscope with an LSM 710 scan head. Due to refinements of the light path within the scan head, the system has better detection capabilities that improve the signal-to-noise ratio of fluorescent images. In addition, the filter-free spectral detection allows the user to define emission collection parameters, rather than being limited to a particular set of installed emission filters.
This instrument has a number of distinct advantages for multiphoton imaging, including a redesigned microscope stand that has increased objective travel, allowing the use of a new 20x high numerical aperture objective with increased transmission in the infrared. An important feature of this microscope is the addition of a low noise photomultiplier tube placed immediately adjacent to the objective for optimal light capture.
The 710 can also be used with a highly sensitive GaAsP detector, which excels at collecting images of fluorescently labeled cells deep within tissue. This improvement has been particularly critical for in vivo applications, and has allowed users to monitor cell motility in intact preparations. Because the GaAsP detector is extremely sensitive to ambient light, we have constructed a light-tight enclosure around this system, so that both microscopes can be used simultaneously.
A near-infrared tunable pulsed femtosecond Ti:Sapphire laser (Chameleon Ultra II) is positioned on the same table as the 510 and 710, allowing the two systems to share the infrared beam from this laser. The IR laser of the two-photon system facilitates imaging of thick specimens, and is capable of imaging of fluorescent dyes or proteins in vivo.
The third confocal system is a custom-built two-photon imaging platform based on a design developed by Dr. Karel Svoboda at HHMI/Janelia Research Campus that is specialized for in vivo imaging. This system also uses a Chameleon Ultra II two-photon laser for excitation and is equipped with a blue LED for epifluorescence imaging. This system offers capabilities beyond that of the commercial Zeiss microscopes in the Core, including simultaneous two-color imaging, more rapid scanning (improved by a factor of two), and greater flexibility due to the open source MATLAB code used to control the instrument. In addition to a standard stereotaxic frame, the system includes a platform for in vivo imaging of both the dorsal horn and dorsal root ganglia of intact spinal cord.
Custom, interchangeable temperature-controlled stages are available for use in live cell/tissue imaging and computer controlled multisite recording. Tissue superfusion is achieved using a gravity-fed system that passes through computer-controlled solenoid valves. Solutions can be oxygenated and, if necessary, heated using an in-line heater or a peltier chamber heater, depending on the application.
The LSM 510 and 710 microscopes are also equipped with DIC optics to allow visualization of cells in brain slices, and imaging can be performed using a black-and-white CCD camera or using laser illumination and a transmitted light detector. With the assistance of Zeiss, we have developed a custom macro which enables mapping of receptor density using two-photon mediated photolysis. An adjustable, heated platform for immobilizing the head or spinal cord of animals during imaging, administering anesthesia, and maintaining animal body temperature is available for use on any of the imaging systems.
The Core offers a wide range of objectives (5x, 10x, and 20x air; 25x multi-immersion; 40x, 63x, and 100x oil; 20x, 40x and 63x water immersion). In addition, we have a piezoelectric focus drive for rapid z-focus control. Piezoelectric positioning drives provide higher focusing speed (~10 vs 100 msec) and better resolution (~1 vs. 100 nm) than stepper motor-drives. This is especially advantageous for applications such as tracking mitochondrial movement and assessing process motility of labeled cells in vivo, where the ability to assess temporal changes is limited by the time required to construct high-quality z-stack images.
Whole cell current and voltage clamp recordings can be performed on either system using an Axon Instruments Multiclamp 700B amplifier. Acquisition is controlled by a Digidata 1322A digital-to-analog converter, and a Pentium 4 PC computer running pClamp10 and Origin analysis software. Additional equipment such as a secondary amplifier (Brownlee), a timer/stimulator (A.M.P.I), black-and-white monitor, camera controller, and halogen power supply are positioned in a moveable rack with castors, allowing this system to be moved into position near either microscope, depending on the needs of the investigator.
The Zeiss Cell Observer system consists of an AxioObserver inverted microscope equipped with fluorescent and transmitted light, an Axiocam MRm, and filter sets compatible with DAPI, GFP, Cy3, and Cy5. An environmental chamber is mounted on the microscope to facilitate control of temperature, humidity, and CO2, allowing maintenance and imaging of live cells over long periods of time. A motorized stage facilitates the identification and imaging of multiple regions of interest. Zen for Widefield software permits flexible design of experiments and provides various post-processing capabilities. The system can also be used to image fixed slides. One feature that is useful to many labs is the motorized stage, which supports acquisition of tiled images and allows users to capture large regions of interest at high magnification. Post-processing features are available to remove line artifacts that are often apparent in such tiled images.
Our Zeiss Axio Zoom microscope provides the advantages of a stereomicroscope – zoom optics and long working distance – with the high resolution of a light microscope. The microscope combines a 16x zoom with a high numerical aperture of 0.25, and the motorized zoom allows quick and easy switching through the entire 16x zoom range without changing objectives.
The microscope is equipped with both fluorescent and transmitted light. Investigators can view and image large samples with excellent brightness and resolution without the need for tiling and stitching. In addition, the long working distance means that the microscope can aid in the dissection of small tissue samples.
The facility has two dedicated image analysis workstations each running Imaris software (Bitplane) for performing 3D reconstruction, isosurface rendering, filament tracing/particle tracking, and quantitative co-localization. The Imaris suite of programs has been designed to accept images in many different formats, include Tiff series, BMP series, and images collected with Leica, Olympus, and Zeiss acquisition software. The available modules are Imaris suite, ImarisXT, Filament tracer, and AutoDeblur. AutoDeblur is a deconvolution software package that produces extremely high-quality results through image restoration. ImarisXT allows users to develop their own task-oriented algorithms for Imaris, or use analysis modules developed by others.
Additional image analysis capabilities are provided by our Neurolucida Neuron Tracing system (Microbrightfield). This system offers a variety of drawing/analysis options, including cell tracing/morphology quantification, 3D brain mapping, and serial section reconstruction. The software is paired with a Zeiss AxioImager microscope equipped with transmitted light and a motorized stage, for tracing directly from slides. Alternatively, image files collected on another system can be imported into Neurolucida. The analysis capabilities of Neurolucida complement those which can be achieved using the Imaris suite of programs, because the latter is geared almost entirely toward analysis of fluorescent images, whereas Neurolucida excels at reconstruction and analysis of brightfield images (e.g. DAB labeled cells). Because this system includes a color camera, it also provides a route for obtaining color images of brightfield samples, such as LacZ.
The Angle2 stereotaxic system (Leica) consists of a software-assisted stereotaxic instrument that facilitates the accurate targeting of brain regions by using software combined with transponders that detect the position of the pipette carrier. Advantages include an on-screen atlas to facilitate identification of targets, the ability to use an angled approach without the need for path-of-approach calculation, and the ability to automatically correct for head tilt.
A MakerBot Replicator 2x allows individuals to use software to generate a 3D model of their desired product; this is then converted into file formats the MakerWare software can output to the 3D printer. This equipment permits inexpensive and efficient production of a wide variety of research components.
Researchers wishing to use the Multiphoton Core must first complete the Application for Service and forward to the Core Manager, Michele Pucak (firstname.lastname@example.org). Once approved, arrangements for an orientation session will be made. Access to the Core will not be granted without an approved application and all users must complete an orientation session.
Procedures for reserving equipment will be reviewed in an orientation session required prior to equipment access. Upon completion of the orientation session and a preliminary training period, equipment is also available on a walk-in basis. Links to reservations calendars are below.
Dwight Bergles, Ph.D.
MPI Core Director
Alex Kolodkin, Ph.D.
Michele Pucak, Ph.D.